There is a baby in the bath water: AcrB contamination is a major problem in membrane-protein crystallization.
In the course of a crystallographic study of the Methanosarcina mazei CorA transporter, the membrane protein was obtained with at least 95% purity and was submitted to crystallization trials. Small crystals (<100 microm) were grown that diffracted to 3.42 A resolution and belonged to space group R32, with unit-cell parameters a = b = 145.74, c = 514.0 A. After molecular-replacement attempts using available CorA structures as search models failed to yield a solution, it was discovered that the crystals consisted of an Escherichia coli contaminating protein, acriflavine resistance protein B (AcrB), that was present at less than 5% in the protein preparations. AcrB contamination is a major problem when expressing membrane proteins in E. coli since it binds naturally to immobilized metal-ion affinity chromatography (IMAC) resins. Here, the structure is compared with previously deposited AcrB structures and strategies are proposed to avoid this contamination.
Veesler
David
D
Architecture et Fonction des Macromolécules Biologiques, CNRS et Universités d'Aix-Marseille I et II, UMR 6098, Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9, France. david.veesler@afmb.univ-mrs.fr
Blangy
Stéphanie
S
Cambillau
Christian
C
Sciara
Giuliano
G
D003461:Crystallography / Q000379:methods / Q000592:standards; D004340:Drug Contamination; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000032:analysis*
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli